Small molecule activators of the mitochondrial protease ClpP induce senescence in triple-negative breast cancer cells and sensitize cells to the Bcl-2 inhibitor venetoclax

ONC201 is a first-in-class, FDA-approved small molecule activator of the mitochondrial ATP-dependent caseinolytic peptidase P (ClpP). This and other related small molecules referred to as ClpP agonists, exert antiproliferative effects in several cancer cell types. We report that ONC201 and highly potent second generation ClpP agonists (TR-57, TR-107), promote induction of senescence in triple-negative breast cancer (TNBC) cell lines. Senescence was determined by increased β-galactosidase (β-gal) activity, downregulation of phosphorylated Rb, c-Myc (Myc), and lamin B1, upregulation of senescent-associated secretory phenotype (SASP), and extended cell proliferation assays. These responses were not observed in ClpP knockout cell lines, demonstrating ClpP-dependence. Proteomics analyses identified multiple events related to the development of senescence including cell cycle arrest and mitochondrial dysfunction. Flow cytometry confirmed an S-phase arrest and DNA damage was detected by Comet assay, 53BP1, phospho-S*Q, and γH2A.X immunostaining. In parallel with this, activation of the ATM pathway and phosphorylation of Chk2 was observed. We determined that ClpP agonist-induced senescence was irreversible in both in vitro and in vivo studies. Following TR-57 treatment and drug washout, cells remained growth arrested which coincided with loss of mitochondrial membrane potential and ability to produce ATP by oxidative phosphorylation. β-gal staining after TR-57 treatment and drug washout demonstrated a sustained increase in β-gal activity, indicating cells are senescent after drug washout. This response was reproduced in vivo wherein senescent 4T1-Luc cells did not develop tumors following injection into mice. Finally, the combination of a ClpP agonist with a known senolytic (venetoclax), synergistically increased the amount of cell death observed. In summary, we show that ClpP agonists stably induce an irreversible senescence in a ClpP-dependent manner that synergizes with venetoclax in TNBC cells.